Process for bio synthesis of nano arsenic trioxide and its use in treatment of diseases including cancer

ABSTRACT

The present invention is a process for bio synthesis of nano arsenic trioxide defined by its low toxicity, higher bio availability and nano particle size with the aid of buttermilk, goat urine,  dolichos biflorous  and other plant materials such as ginger,  momordica charantia  and  musa paradisiaca . The invention is carried out in different steps involving purification of crude form of arsenic trioxide by boiling it with buttermilk, goat urine and extract of  dolichos biflorous  in subsequent steps, followed by the trituration of the bio purified arsenic trioxide with extracts of ginger and  momordica charantia  in subsequent steps and heating of the dry product obtained after trituration with  musa paradisiaca  resulting in the production of novel nano arsenic trioxide. The product is effective in the treatment of various diseases including different types of cancer in animals and humans. The product obtained through the process is less toxic with higher bio availability.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of PCT application numberPCT/IB2012/054992, filed Sep. 20, 2012.

FIELD OF INVENTION

The present invention is a novel process for production of nano arsenictrioxide with the aid of plant materials, buttermilk and goats urine.The process is environment friendly. The novel product obtained throughthe present inventive process is more bio available and therapeuticallyeffective in the treatment of various diseases including but not limitedto leukemia, other types of malignant and benign tumours, rheumatism.The present inventive product also finds application in agriculture,horticulture, veterinary treatment and other fields where arsenictrioxide can be used as known to people skilled in the art.

BACKGROUND OF INVENTION

Applicability of Arsenic minerals is known in traditional Chinese andIndian medicine since ancient times. In the 18th Century Thomas Fowlercompounded a potassium bicarbonate based solution of arsenic trioxide(As₂O₃) which came to be known as Fowler's solution. Pharmacology textsof the 18th Century indicate the use of arsenical pastes for thetreatment of variety of diseases including cancer [Karen H. Antman;Introduction: The history of arsenic trioxide in cancer therapy; TheOncologist 2001; 6(suppl 2):1-2]. In the 1990's it was reported by theChinese investigators that herbal mixture containing arsenic trioxidecould induce complete remission in patients suffering from acutepromyelocytic leukaemia. [Dan Douer, Wendy Hu et. Al.; Arsenic trioxide(Trisenox) Therapy for Acute Promyelocytic Leukaemia in the setting ofhematopoietic stem cell transplantation; The Oncologist 2003;8:132-140].

Arsenic widely exists in nature in its trivalent and pentavalent forms.Arsenic toxicity highly depends on its chemical form. In Indian andChinese traditional medicines three forms of arsenic minerals i.e.,Orpiment, Realgar and Arsenolite, are used alone and also in conjunctionwith other minerals for treatment of various diseases. The dispositionof these arsenicals in the body depends on various key factors,including solubility, absorption, distribution, and excretion. Arsenictrioxide is the most bio available but it is highly toxic compared toOrpiment and Realger. Realgar is also a major component in bhasmas ofIndian Ayurvedic medicine. Realger is used both for external as well asinternal application. Arsenic trioxide, obtained after purification ofArsenolite, has short term toxicity due to which its therapeuticapplication is a major concern. To increase the therapeutic applicationof Realgar, nano particles of Realgar is prepared by cryogrinding withpolyvinylpyrrolidone and Sodium Dodecyl Sulphate. (Jie Liu et. al.,Mineral Arsenicals in Traditional Medicines:Orpiment, Realger, andArsenolite, The Journal of Pharmacology and experimental therapeutics,Vol. 326, No. 2, pg. 363-368, 2008).

Prior art search indicates that arsenic trioxide is and has been used inthe treatment of cancer. EP2374463, EP2394702, EP 1562616, WO9924029,U.S. Pat. No. 6,884,439, U.S. Pat. No. 6,855,339, U.S. Pat. No.6,982,096, U.S. Pat. No. 6,723,351, U.S. Pat. No. 6,720,011 are theresults of prior art search indicating use of arsenic trioxide intherapy. However the present invention is unique in terms of the processof preparation and the novel nano arsenic trioxide obtained through thepresent inventive process is generally of particle size ranging from 10nm to 1000 nm. Further, the novel product is chemically more stable, bioavailable and safe due to less toxic nature for therapeuticadministration in humans, animals and plants. The prior art process forproduction of arsenic trioxide that is used for therapeutic applicationinvolves purification of arsenic trioxide through chemical process. Thepresent invention purifies arsenic trioxide through bio synthesis.

The present invention relates to the production of novel nano arsenictrioxide by a novel process, which involves purification, making itchemically more stable, and particle size reduction of arsenic trioxide,with plant materials, buttermilk and goat urine. The plant materialsused in the present process are Dolichos biflorous, Momordica charantia,Zingiber officinale, Musa paradisica. The scope of the invention alsoinclude use of the active ingredients found in the herein mentionedplant materials, buttermilk, goat urine or other plant, organic orinorganic materials, containing cellulose, with similar characteristicsand chemical properties, as known to the people skilled in the art, forthe purpose of inventive process and production of the novel product.

Buttermilk is a fermented dairy product obtained from cow's milk. Thetartness of buttermilk is due to the presence of lactic acid. The pHlevel of buttermilk is generally noted to be 4.41 to 4.84. Butter milkcan be made through traditional process, by culturing and also throughacidification process of milk. One phase of the invention involvespurification of arsenic trioxide with the aid of buttermilk. Use ofbuttermilk is one embodiment of this invention and it is possible toderive the same results if buttermilk is substituted with any otherfermented form of milk either obtained through traditional methods orcultured. The substitution of fermented milk obtained throughtraditional methods or cultured with that of chemically produced orinduced active ingredients with same effect known to people skilled inthe art cannot be ruled out.

Goat and Cow urine have been an ingredient in Indian Ayurvedic medicineand other traditional Indian Medicine. Goat Urine is used in treatmentof tuberculosis by tribals in Western ghats, India [P. Padmanabhan &amp; K. A. Sujana, 2008, Animal products in traditional medicine fromAttapady hills of Western Ghats, Indian Journal of Traditional Medicine,Vol. 7(2), pg. 326-329]. However, the use of the goat urine as in thepresent process is not traditional knowledge or anticipated.

The medicinal values of plant materials belonging to genus Zingiber andfamily Zingiberaceae is well known in traditional medicines. ZingiberOfficinale is plant species of genus Zingiber. The rhizome of ZingiberOfficinale, Ginger, is one of the most widely used species of gingerfamily Zingiberaceae and is known for its medicinal use in traditionalChinese and Indian medicine. Ginger has many active ingredients such asSesquiterpene hydrocarbons predominantly Zingiberene, active gingerolsthat can be converted to shogaols, Zingerone, Paradol. 6-Gingerol and6-shogaols have shown pharmacological activities including anti pyretic,analgesic and in treatment of chemotherapy induced nausea (Monograph,Alternative Medicine Review, Volume 8, No. 3, 2003, pg. 331-335).However, use of ginger or extract of plant material of family ofZingiber Officinale for particle size reduction of metalloid is notknown. The present inventive process also involve particle sizereduction of arsenic trioxide to nano particles with aid of ginger.

Momordica charantia (Bitter Melon) is a tropical vegetable of familyCucurbitaceae. It is a flowering vine with known medicinal properties.Its fruits contain glycosides, saponins, alkaloids, reducing sugars,resins, phenolic constituents, fixed oils and free acids. Chemicalconstituents in Momordica Charantia are Alkaloids, charantin, charine,cryptoxanthin, cucurbitins, cucurbitacins, cucurbitanes, cycloartenols,diosgenin, elaeostearic acids, erythrodiol, galacturonic acids, gentisicacid, goyaglycosides, goyasaponins, guanylate cyclise inhibitors,gypsogenin, hydroxytryptamines, karounidiols, lanosterol, lauric acid,linoleic acid, linolenic acid, momorcharasides, momorcharins,momordenol, momordicilin, momordicins, momordicinin, momordicosides,momordin, momordolo, multiflorenol, myristic acid, nerolidol, oleanolicacid, oleic acid, oxalic, acid, pentadecans, peptides, petroselinicacid, polypeptides, proteins, ribosome-inactivating proteins, rosmarinicacid, rubixanthin, spinasterol, steroidal glycosides, stigmasta-diols,stigmasterol, taraxerol, trehalose, trypsin inhibitors, uracil, vacine,v-insulin, verbascoside, vicine, zeatin, zeatin riboside, zeaxanthin,zeinoxanthin Amino acids-aspartic acid, serine, glutamic acid, thscinne,alanine, g-amino butyric acid and pipecolic acid, ascorbigen,b-sitosterol-d-glucoside, citrulline, elasterol, flavochrome, lutein,lycopene, pipecolic acid.

Musa paradisica is a herbaceous plant of the genus Musa. The olderscientific name Musa paradisica is seldom used due to the advent ofhybrid varieties. Presently the species are referred to as Musaacuminata or Musa balbisiana or triploid hybrids. The fruit as well asthe plant materials have medicinal value. In the present invention theextract of the plant material is used.

Dolichos biflorous is commonly used as an astringent, diuretic andtonic. The enzymes found in Dolichos biflorous have been identified asUrease, Allantoinase, Ribonuclease, Nicotinamide deaminase,b-n-acetylglucosaminidase, a and b galactosidase, a-mannosidase and bglucosidase.

SUMMARY OF THE INVENTION

To increase the therapeutic applicability and bio availability ofarsenic trioxide, the present invention purifies, through biopurification, arsenic trioxide available in the market by the process ofboiling, trituration, and heating, with plant materials, buttermilk andgoats urine. In short, the arsenic trioxide in its crude form asavailable in the market is put through process of purification byboiling with buttermilk, Goat urine and Dolichos biflorous inconsecutive steps. Thus purified arsenic trioxide is put throughparticle size reduction by process of trituration with extract of plantmaterial of taxonomical genus Momordica and extract of rizhome oftaxonomical genus Zingiber. The product obtained from trituration isthen heated with extract of plant material of genus Musa. The process ofheating is carried out in a container with closed lid so as to collectthe sublimed product. The product collected from the inner side of thelid of the container is the novel nano arsenic trioxide with hightherapeutic value and solubility. Further, bioavailability andtherapeutic applicability of the novel product, nano arsenic trioxide,obtained through this inventive process is higher due to its less toxicnature and particle size.

DETAILED DESCRIPTION

Arsenic trioxide, crude powder form, available in the market, is biopurified with the aid of butter milk, Goat urine, and aqueous extract ofDolichos biflorous in various steps followed by trituration and heatingas detailed below:

Step 1: Arsenic trioxide in coarse powder form is submerged into anearthen vessel containing boiling butter milk. The arsenic trioxide ishung to submerge into the boiling butter milk without touching sides ofthe earthen vessel. This process is carried out for seven hours. At alltimes the arsenic trioxide should be submerged during the process ofboiling. Hence, buttermilk will have to be added repeatedly during theprocess of boiling to maintain arsenic trioxide in submerged conditionthroughout the period.

Step 2: After boiling arsenic trioxide with butter milk in accordancewith step 1, the same is hung and submerged into boiling goat urinecontained in an earthen vessel, without touching the sides of thecontainer. This process is carried out for seven hours. During this steptoo arsenic trioxide is to be retained submerged through the wholeperiod of boiling with goat urine and hence it would be required to addgoat urine during the process to maintain the arsenic trioxide insubmerged condition.

Step 3: After boiling arsenic trioxide with goat urine in accordancewith step 2, the same is then hung and submerged, into boiling aqueousextract of Dolichos biflorous contained in an earthen vessel, withouttouching the sides of the container. This process is carried out for 7hours. Similar to the earlier steps, addition of aqueous extract ofDolichos biflorous repeatedly is required throughout the period ofboiling to maintain the arsenic trioxide completely submerged.

The time of seven hours for the process continuation provided in step 1to 3 cannot be considered as limiting. In the process arsenic trioxidecan be hung using a cotton string or with materials of similarcharacteristic. The vessel used can be an earthen container or anyvessel with similar characteristics that may be known to persons skilledin the art. Product is yellowish white powder. Biological variation thatmay occur in goat urine, buttermilk, and the extract of plant materialsis anticipated in the present invention. The scope of the presentinvention also anticipates the use of buttermilk, goat urine and extractof dolichos biflorous in interchangable order, as in similar result ispossible if the Arsenic Trioxide is put through step 1, 2 or 3 indifferent orders where by step 3 or step 2 can be performed as step 1 orvice versa.

Step 4: The product of Step 3 is triturated with extract of Momordicacharantia for 7 hours. The time for trituration cannot be consideredlimiting. The process of trituration is repeated thrice consecutively,i.e., once the product of trituration is completely dry then the nexttrituration should be commenced with. Generally it takes 7 hours for themixture to dry out but 7 hours cannot be considered limiting, as thenumber of hours depends upon the quantity of the material.

Step 5: The product of Step 4 is triturated with extract of ginger,rhizome of Zingiber officinale for 7 hours. The time for triturationcannot be considered limiting. Similar to Step 4, the trituration iscarried out thrice consecutively whereby the dry product obtained aftertrituration is further triturated with extract of rhizome of ZinziberOfficianale until dry.

The time provided for trituration i.e., seven hours cannot be consideredlimiting and the desired result can be obtained if the trituration iscarried out for less or more hours. The particle size of the productwould vary with the number of hours of trituration. The possibility ofobtaining the desired result if the trituration is carried out with amixture of the plant materials or its active ingredients, specified insteps 4 and 5, or other organic or inorganic materials with similarcharacteristics, is within the scope of this invention. The resultantproduct is generally yellowish white in colour.

Step 6: The product of Step 5 is heated with extract of the stem of Musaparadisica in a closed earthen container with removable lid, volumeratio of the product and extract of the stem of Musa paradisica ispreferably 1:10. The heating is gradual and carried out for 12 hours.The heating duration cannot be considered limiting. The sublimatedproduct on the inner side of the lid of the container is the finalproduct. The final product is generally white in colour with mild yellowtinge. The maximum temperature to which the present process is subjectedto is approximately 500 degree centigrade. However, the desirable resultcan be obtained at below or above 500 degree centigrade and hence thetemperature of heating cannot be considered as limiting. The temperatureat which the product is obtained would vary with the quantity of thecrude arsenic trioxide and other ingredients.

The container or apparatus for carrying out the present process is anearthen vessel with a removable lid or any other vessel, which is knownto the people skilled in the art to bring about similar results.

The ingredients in the steps mentioned above are one embodiment of theinvention. The scope of the invention also include active ingredients ofthe plant and other materials used in the process, organic as well asinorganic, or other plant materials and organic or inorganic materialswith similar characteristics or chemical properties, containingcellulose, that are known to people skilled in the art to bring desiredeffects on substitution of buttermilk, Goat Urine, Dolichos biflorous,Momordica Charantia, Ginger, Musa Paradisica or other aids in this novelprocess for production of nano arsenic trioxide.

The administration of this chemically more stable, novel nano arsenictrioxide obtained through this inventive process can be done as is or incombination with other minerals, metals, chemical elements or compounds.Both in vitro and in vivo administration is possible with this novelnano arsenic trioxide. The oral administration of the product can be inconjunction with honey or water or any other suitable carrier. Thedosage of administration can range from 0.01 mg per kg body weight to 10mg per kg body weight. It can be administered on daily basis dependingon the nature of the disease. In case of cancer the period ofadministration can be until complete remission or reduction of tumour asthe case may be.

The dosage form can be in powder, tablet, capsule, caplet, effervescent,fluid, gelatinous, granules or in any other palatable and administrableform.

The nano arsenic trioxide obtained through this process, generally ofsize varying from 10 nm to 1000 nm, can also be used in nutraceutical,herbal and mineral composition apart from pharmaceutical or othertherapeutic composition.

The product of the present inventive process is also effective intreatment of cancers which are malignant and benign, haematologicalmalignancies such as Promyelocytic Leukemia, other types of Leukemias,Lymphomas, solid tumours like Lung Cancers, Liver cancers etc. Theapplication of this Nano Arsenic Trioxide is not only limited to variouscancers but also includes other degenerative and metabolic disorders ofvital organs such as lungs, liver, brain, kidneys etc. This has wideapplication in Neurological cancer disorders and also in multipleinfective disorders especially but not limiting to antibiotics or antimicrobial resistant infections such as Tuberculosis. This novel arsenictrioxide has got specific action on various enzymatic and hormonalactivities in the body for treatment of diseases, including but notlimited to, Diabetes. It has got specific action on bone developments ingeneral and at epiphyseal level including prevention and cure of boneand other types of cancers. Use of the present novel product in thetreatment of animals, plants, herbs or other flora and industrialapplication in nutraceutical, cosmetic, herbal, mineral and othercomposition is anticipated.

The following are a few examples. However the examples providedhereunder must not be considered limiting as to the scope, working oruse of the present invention:

Examples of Formulation

Actual Arsenic Weight of Total weight of Trioxide lactose Magnesiumweight, powder to dose monohydrate stearate in in mg/ be filled in Sr.No. in mg in mg mg capsule mg/capsule 1 6 57.00 0.5 63.50 64* 2 12 56.000.5 68.50 69*

What is claimed is:
 1. The process comprising the steps of: preparing afirst product by submerging and boiling crude powder form of arsenictrioxide subsequently in each of the following: buttermilk, Goat urineand aqueous extract of dolichos biflorus; performing a first triturationof the prepared first product with an extract of Momordica charantiauntil dry; performing a second trituration of the dry product producedafter the first trituration with extract of ginger until dry; andheating a resultant product obtained after the second trituration withan extract of Musa paradisiaca in an apparatus with a lid and collectinga resultant sublimed product comprising nano arsenic trioxide, from aninner side of the lid.
 2. The process of claim 1, wherein the resultantsublimed product obtained is palatable and administrable.
 3. The productof the process of claim 1.